Children with Hirschsprung disease exhibited alterations in host-microbial co-metabolism after pull-through operation.

PURPOSE: This study aims to compare the fecal metabolome in post pull-through HD with and without HAEC patients and healthy young children using nuclear magnetic resonance (NMR) spectroscopy. METHODS: Fresh fecal samples were collected from children under 5 years of age in both post-pull-through HD patients and healthy Thai children. A total of 20 fecal samples were then analyzed using NMR spectroscopy. RESULTS: Thirty-four metabolites identified among HD and healthy children younger than 5 years were compared. HD samples demonstrated a significant decrease in acetoin, phenylacetylglutamine, and N-acetylornithine (corrected p value = 0.01, 0.04, and 0.004, respectively). Succinate and xylose significantly decreased in HD with HAEC group compared to HD without HAEC group (corrected p value = 0.04 and 0.02, respectively). Moreover, glutamine and glutamate metabolism, and alanine, aspartate, and glutamate metabolism were the significant pathways involved, with pathway impact 0.42 and 0.50, respectively (corrected p value = 0.02 and 0.04, respectively). CONCLUSION: Differences in class, quantity, and metabolism of protein and other metabolites in young children with HD after pull-through operation were identified. Most of the associated metabolic pathways were correlated with the amino acids metabolism, which is required to maintain intestinal integrity and function.

Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

The Hallmarks of Liver Fluke Related Cholangiocarcinoma: Insight into Drug Target Possibility.

Cholangiocarcinoma (CCA) is a malignant tumor of the biliary tree that is classified into three groups based on its anatomic location: intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA). Perihilar CCA is the most common type and accounts for 50-60% of CCA cases. It is followed by distal CCA and then intrahepatic CCA that account for 20-30% and 10-20% of cases, respectively. This chapter discusses the hallmarks of liver fluke related CCA and explores insights into drug target possibilities.

Modification of the AJCC/UICC 8th edition staging system for intrahepatic cholangiocarcinoma: proposal for an alternative staging system from cholangiocarcinoma-prevalent Northeast Thailand.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) arises from bile ducts within the liver. Thailand has the highest incidence of CCA worldwide, with a high mortality rate. Early diagnosis and accurate prognostic stratification can improve overall survival. We aim to modify the AJCC/UICC 8th edition staging system for iCCA by creating the Khon Kaen University (KKU) staging system for more precise patient stratification and prognostic prediction. METHODS: A total of 298 iCCA patients who underwent hepatectomy were included in this retrospective study at the Srinagarind Hospital, Khon Kaen University, Thailand. Univariate and multivariate analysis were performed to examine survival rate, hazard ratio, and prognostic factors. RESULTS: Univariate and multivariate analysis of the cohort showed that growth patterns, histological type, histological grade, lymph node metastasis and distant metastasis were independent prognostic factors when compared to the respective reference groups. The 8th AJCC staging system incorporated growth patterns into the KKU staging system. This model modified AJCC stages I, II, and III for better prediction of patient survival. CONCLUSION: Growth patterns were incorporated to improve the 8th AJCC staging system for prognostication of iCCA patients in Northeast Thailand. We propose the KKU staging system as an alternative model for iCCA staging to augment the accuracy of survival prognostication.

Overexpression of a panel of cancer stem cell markers enhances the predictive capability of the progression and recurrence in the early stage cholangiocarcinoma.

BACKGROUND: Cancer recurrence is the important problem of cholangiocarcinoma (CCA) patients, lead to a very high mortality rate. Therefore, the identification of candidate markers to predict CCA recurrence is needed in order to effectively manage the disease. This study aims to examine the predictive value of cancer stem cell (CSC) markers on the progression and recurrence of CCA patients. METHODS: The expression of 6 putative CSC markers, cluster of differentiation 44 (CD44), CD44 variant 6 (CD44v6), CD44 variants 8-10 (CD44v8-10), cluster of differentiation 133 (CD133), epithelial cell adhesion molecule (EpCAM), and aldehyde dehydrogenase 1A1 (ALDH1A1), was investigated in 178 CCA tissue samples using immunohistochemistry (IHC) and analyzed with respect to clinicopathological data and patient outcome including recurrence-free survival (RFS) and overall survival (OS). The candidate CSC markers were also investigated in serum from CCA patients, and explored for their predictive ability on CCA recurrence. RESULTS: Elevated protein level of CD44 and positive expression of CD44v6 and CD44v8-10 were significantly associated with short RFS and OS, while high levels of ALDH1A1 were correlated with a favorable prognosis patient. The elevated CD44v6 level was also correlated with higher tumor staging, whereas a decreasing level of ALDH1A1 was correlated with lower tumor staging. The levels of CD44, CD44v6 and CD44v8-10 were also correlated and were associated with a poor outcome. Furthermore, soluble CD44, CD44v6, CD44v8-10 and EpCAM were significantly increased in the recurrence group for early stage CCA; they also correlated with high levels of the tumor marker CA19-9. Elevated levels of CD44, CD44v6, CD44v8-10 or EpCAM alone or in combination has the potential to predict CCA recurrence. CONCLUSIONS: The overexpression of CD44, CD44v6, CD44v8-10 and EpCAM increases predictability of post-operative CCA recurrence. Moreover, the overexpression of the panel of CSC markers combined with CA19-9 could improve our predictive ability for tumor recurrence in early stage CCA patients. This result may be beneficial for the patients in order to predict the outcome after treatment and may be useful for clinical intervention in order to improve patient survival.

A panel of protein kinase high expression is associated with postoperative recurrence in cholangiocarcinoma.

BACKGROUND: Cancer recurrence is one of the most concerning clinical problems of cholangiocarcinoma (CCA) patients after treatment. However, an identification of predictive factor on Opisthorchis viverrini (OV)-associated CCA recurrence is not well elucidated. In the present study, we aimed to investigate the correlation of twelve targeted protein kinases with CCA recurrence. METHODS: Twelve protein kinases, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2, 3, 4 (HER2, HER3, HER4), vascular endothelial growth factor receptor 3 (VEGFR3), vascular endothelial growth factor-C (VEGF-C), erythropoietin-producing hepatocellular carcinoma receptor type-A3 (EphA3), EphrinA1, phosphor-serine/threonine kinase 1 (p-Akt1), serine/threonine kinase 1 (Akt1), beta-catenin and protein Wnt5a (Wnt5a) were examined using immunohistochemistry. Pre-operative serum tumor markers, CA19-9 and CEA were also investigated. RESULTS: Among twelve protein kinases, EGFR, HER4, and EphA3 were associated with tumor recurrence status, recurrence-free survival (RFS) and overall survival (OS). Multivariate cox regression demonstrated that EGFR, HER4, EphA3 or the panel of high expression of these proteins was an independent prognostic factor for tumor recurrence. The combination of high expression of these proteins with a high level of CA19-9 could improve the predictive ability on tumor recurrence. Moreover, the patients were stratified more accurately when analyzed using the combination of high expression of these proteins with primary tumor (T) or lymph node metastasis (N) status. CONCLUSION: EGFR, HER4, EphA3 or the panel of high expression of these proteins is an independent prognostic factor for post-operative CCA recurrence.

A comparison of the proportion of early stage cholangiocarcinoma found in an ultrasound-screening program compared to walk-in patients.

BACKGROUND: Patients with cholangiocarcinoma (CCA) usually have no specific symptoms until an advance stage of the disease and curative treatment is not possible. Patients with early stage, operable disease can be found using ultrasonography (US). A US-screening program was implemented in Thailand where CCA incidence is the highest worldwide. Here we evaluate the effectiveness of the program by comparing the proportion of individuals with early stage CCA in the screening group with that of the walk-in group presenting at hospitals with clinical symptoms. METHODS: All patients had a pathological diagnosis of CCA. The difference in the proportions and the 95% confidence interval (CI) were obtained using binomial regression. RESULTS: Of the 762 histologically proven CCA cases, 161 were from the screening group and 601 from the walk-in group. The proportion of early stage CCA (stages 0 to II) diagnosed was 84.5% in the screening and 21.6% in the walk-in groups. After adjustment age, gender, and liver fluke infection, there was a significantly higher proportion (P < 0.001) and higher chance (P < 0.001) of having early stage CCA in the screening group than in the walk-in group. CONCLUSIONS: US-screening is an effective tool for detecting early stage, operable CCA in high incidence areas.

Discovery and Qualification of Serum Protein Biomarker Candidates for Cholangiocarcinoma Diagnosis.

Cholangiocarcinoma (CCA) is a major health problem in northeastern Thailand. The majority of CCA cases are clinically silent and difficult to detect at an early stage. Although abdominal ultrasonography (US) can detect premalignant periductal fibrosis (PDF), this method is not suitable for screening populations in remote areas. With the goal of developing a blood test for detecting CCA in the at-risk population, we carried out serum protein biomarker discovery and qualification. Label-free shotgun proteomics was performed on depleted serum samples from 30 participants (n = 10 for US-normal, US-PDF, and CCA groups). Of 40 protein candidates selected using multiple reaction monitoring on 90 additional serum samples (n = 30 per group), 11 discriminatory proteins were obtained using supervised multivariate statistical analysis. We further evaluated 3 candidates using ELISA and immunohistochemistry (IHC). S100A9, thioredoxin (TRX), and cadherin-related family member 2 (CDHR2) were significantly different between CCA and normal, and CCA and PDF groups when measured in an additional 247 serum samples (P < 0.0001). By IHC, TRX and CDHR2 were detected in the cytoplasm and nucleus of CCA and inflammatory cells. S100A9 was detected in the infiltrating tumor stroma immune cells. Proteomics discovery and qualification in depleted sera revealed promising biomarker candidates for CCA diagnosis.

Dihydroartemisinin induces apoptosis and autophagy-dependent cell death in cholangiocarcinoma through a DAPK1-BECLIN1 pathway.

Cholangiocarcinoma (CCA) is a very aggressive cancer arising from the malignant transformation of cholangiocytes. Intrahepatic CCA is associated with reactive inflammation and intense fibrosis of the hepatobiliary tract. Dihydroartemisinin (DHA), the active compound found in Artemisia annua, has been shown to possess anti-tumor activity in a variety of human cancers, including hepatoma. Here, we tested the ability of DHA to specifically kill CCA cells and have investigated the underlying mechanisms. DHA induced both apoptosis and autophagy-dependent caspase-independent cell death in many CCA cell lines, while being slightly toxic to immortalized cholangiocytes. DHA induced the expression of many apoptosis- and autophagy-related genes in CCA cells. In particular, it greatly induced the expression of DAPK1, and reduced the interaction of BECLIN1 with BCL-2 while promoting its interaction with PI3KC3. Genetic silencing of DAPK1 prevented DHA-induced autophagy. Pharmacologic and genetic inhibition of BECLIN1 function prevented autophagy and cell death induced by DHA in CCA cells. These data unravel a novel pathway of DHA cancer toxicity and open the possibility to introduce DHA in the therapeutic regimen for the treatment of CCA.

Upregulation of transferrin receptor-1 induces cholangiocarcinoma progression via induction of labile iron pool.

Labile iron pool is a cellular source of ions available for Fenton reactions resulting in oxidative stress. Living organisms avoid an excess of free irons by a tight control of iron homeostasis. We investigated the altered expression of iron regulatory proteins and iron discrimination in the development of liver fluke-associated cholangiocarcinoma. Additionally, the levels of labile iron pool and the functions of transferrin receptor-1 on cholangiocarcinoma development were also identified. Iron deposition was determined using the Prussian blue staining method in human cholangiocarcinoma tissues. We investigated the alteration of iron regulatory proteins including transferrin, transferrin receptor-1, ferritin, ferroportin, hepcidin, and divalent metal transporter-1 in cholangiocarcinoma tissues using immunohistochemistry. The clinicopathological data of cholangiocarcinoma patients and the expressions of proteins were analyzed. Moreover, the level of intracellular labile iron pool in cholangiocarcinoma cell lines was identified by the RhoNox-1 staining method. We further demonstrated transferrin receptor-1 functions on cell proliferation and migration upon small interfering RNA for human transferrin receptor 1 transfection. Results show that Iron was strongly stained in tumor tissues, whereas negative staining was observed in normal bile ducts of healthy donors. Interestingly, high iron accumulation was significantly correlated with poor prognosis of cholangiocarcinoma patients. The expressions of iron regulatory proteins in human cholangiocarcinoma tissues and normal liver from cadaveric donors revealed that transferrin receptor-1 expression was increased in the cancer cells of cholangiocarcinoma tissues when compared with the adjacent normal bile ducts and was significantly correlated with cholangiocarcinoma metastasis. Labile iron pool level and transferrin receptor-1 expression were significantly increased in KKU-214 and KKU-213 when compared with cholangiocyte cells (MMNK1). Additionally, the suppression of transferrin receptor-1 expression significantly decreased intracellular labile iron pool, cholangiocarcinoma migration, and cell proliferation when compared with control media and control small interfering RNA. In Conclusion, high expression of transferrin receptor-1 resulting in iron uptake contributes to increase in the labile iron pool which plays roles in cholangiocarcinoma progression with aggressive clinical outcomes.

Inhibition of l-type amino acid transporter 1 activity as a new therapeutic target for cholangiocarcinoma treatment.

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na+-independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [14C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue-stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal's body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that suppression of l-type amino acid transporter-1 activity using JPH203 might be used as a new therapeutic strategy for cholangiocarcinoma treatment.

Establishment of cholangiocarcinoma cell lines from patients in the endemic area of liver fluke infection in Thailand.

Cholangiocarcinoma is a rare type of cancer which is an increasingly discernible health threat. The disease is usually very difficult in diagnosis and various treatment modalities are typically not effective. Cholangiocarcinoma is a complex and very heterogeneous malignancy characterized by tumor location, different risk factors, molecular profiling, and prognosis. Cancer cell lines represent an important tool for investigation in various aspects of tumor biology and molecular therapeutics. We established two cell lines, KKU-452 and KKU-023, which were derived from patients residing in the endemic area of liver fluke infection in Thailand. Both of tumor tissues have gross pathology of perihilar and intrahepatic mass-forming cholangiocarcinoma. Two cell lines were characterized for their biological, molecular and genetic properties. KKU-452 and KKU-023 cells are both adherent cells with epithelium morphology, but have some differences in their growth pattern (a doubling time of 17.9 vs 34.8 h, respectively) and the expression of epithelial bile duct markers, CK7 and CK19. Cytogenetic analysis of KKU-452 and KKU-023 cells revealed their highly complex karyotypes; hypertriploid and hypotetraploid, respectively, with multiple chromosomal aberrations. Both cell lines showed mutations in p53 but not in KRAS. KKU-452 showed a very rapid migration and invasion properties in concert with low expression of E-cadherin and high expression of N-cadherin, whereas KKU-023 showed opposite characters. KKU-023, but not KKU-452, showed in vivo tumorigenicity in xenografted nude mice. Those two established cholangiocarcinoma cell lines with unique characters may be valuable for better understanding the process of carcinogenesis and developing new therapeutics for the patients.

CD44 variant-dependent redox status regulation in liver fluke-associated cholangiocarcinoma: A target for cholangiocarcinoma treatment.

Expression of CD44, especially the variant isoforms (CD44v) of this major cancer stem cell marker, contributes to reactive oxygen species (ROS) defense through stabilizing xCT (a cystine-glutamate transporter) and promoting glutathione synthesis. This enhances cancer development and increases chemotherapy resistance. We investigate the role of CD44v in the regulation of the ROS defense system in cholangiocarcinoma (CCA). Immunohistochemical staining of CD44v and p38(MAPK) (a major ROS target) expression in Opisthorchis viverrini-induced hamster CCA tissues (at 60, 90, 120, and 180 days) reveals a decreased phospho-p38(MAPK) signal, whereas the CD44v signal was increased during bile duct transformation. Patients with CCA showed CD44v overexpression and negative-phospho-p38(MAPK) patients a significantly shorter survival rate than the low CD44v signal and positive-phospho-p38(MAPK) patients (P = 0.030). Knockdown of CD44 showed that xCT and glutathione levels were decreased, leading to a high level of ROS. We examined xCT-targeted CD44v cancer stem cell therapy using sulfasalazine. Glutathione decreased and ROS increased after the treatment, leading to inhibition of cell proliferation and induction of cell death. Thus, the accumulation of CD44v leads to the suppression of p38(MAPK) in transforming bile duct cells. The redox status regulation of CCA cells depends on the expression of CD44v to contribute the xCT function and is a link to the poor prognosis of patients. Thus, an xCT inhibitor could inhibit cell growth and activate cell death. This suggests that an xCT-targeting drug may improve CCA therapy by sensitization to the available drug (e.g. gemcitabine) by blocking the mechanism of the cell's ROS defensive system.

Cohort profile: cholangiocarcinoma screening and care program (CASCAP).

BACKGROUND: Cholangiocarcinoma (CCA) is an extremely aggressive cancer that is usually fatal. Although globally morbidity and mortality are increasing, knowledge of the disease remains limited. The Mekong region of Southeast Asia, and particularly the northeast of Thailand, has by far the highest incidence of CCA worldwide with 135.4 per 100,000 among males and 43.0 per 100,000 among females being reported in Khon Kaen Province. Most patients are first seen during late stage disease with 5-year survival being less than 10%. Starting in 1984, control and prevention strategies have been focused on health education. Although early detection can substantially increase 5-year survival, there are currently no strategies to increase early diagnosis. METHODS/DESIGN: The Cholangiocarcinoma Screening and Care Program (CASCAP) is a prospective cohort study comprising two cohorts- the screening and the patient cohorts. For the screening cohort, ultrasound examination will be carried out regularly at least annually to determine whether there is current bile duct and/or liver pathology so that the optimal screening program for early diagnosis can be established. This cohort is expected to include at least 150,000 individuals coming from high-risk areas for CCA. For the patient cohort, it is estimated that about 25,000 CCA patients will be included during the 5-year recruitment period. All CCA patients will be treated according to routine clinical care and followed so that effective surgical treatment can be formulated. This cohort is indeed a conventional cancer registry. Thus, CASCAP is an ongoing project in which the number of participants changes dynamically. DISCUSSIONS: This is the first project on CCA that involves screening the at risk population at the community level. At the time of preparing this report, a total of 85,927 individuals have been enrolled in the screening cohort, 55.0% of whom have already undergone ultrasound screening, and 2661 CCA cases have been enrolled in the patient cohort. Among the participants of the screening, whose mean age was 53.8±9.8 years, 55.6% were female, 77.5% attained primary school as the highest level of education, 79.9% were farmers, 29.9%, reported having relatives with CCA, 89.1% had eaten uncooked fish, and 42.2% of those who had been tested for liver fluke were found to be infected.

Activated macrophages promote Wnt/ß-catenin signaling in cholangiocarcinoma cells.

The Wnt/ß-catenin signaling pathway is pathologically activated in cholangiocarcinoma (CCA). Here, we determined the expression profile as well as biological role of activated Wnt/ß-catenin signaling in CCA. The quantitative reverse transcription polymerase chain reaction demonstrated that Wnt3a, Wnt5a, and Wnt7b mRNA were significantly higher in CCA tissues than adjacent non-tumor tissues and normal liver tissues. Immunohistochemical staining revealed that Wnt3a, Wnt5a, and Wnt7b were positive in 92.1, 76.3, and 100 % of 38 CCA tissues studied. It was noted that Wnt3 had a low expression in tumor cells, whereas a high expression was mainly found in inflammatory cells. Interestingly, a high expression level of Wnt5a was significantly correlated to poor survival of CCA patients (P=0.009). Membrane localization of ß-catenin was reduced in the tumors compared to normal bile duct epithelia, and we also found that 73.7 % of CCA cases showed the cytoplasmic localization. Inflammation is known to be a risk factor for CCA development, and we tested whether this might induce Wnt/ß-catenin signaling. We found that lipopolysaccharides (LPS) elevated the expression of Wnt3 both mRNA and protein levels in the macrophage cell line. Additionally, the conditioned media taken from LPS-induced activated macrophage culture promoted ß-catenin accumulation in CCA cells. Furthermore, transient suppression of ß-catenin by siRNA significantly induced growth inhibition of CCA cells, concurrently with decreasing cyclin D1 protein level. In conclusion, the present study reports the abundant expression of Wnt protein family and ß-catenin in CCA as well as the effect of inflammatory condition on Wnt/ß-catenin activation in CCA cells. Importantly, abrogation of ß-catenin expression caused significant CCA cell growth inhibition. Thus, the Wnt/ß-catenin signaling pathway may contribute to CCA cell proliferation and hence may serve as a prognostic marker for CCA progression and provide a potential target for CCA therapy.

Increased EphB2 expression predicts cholangiocarcinoma metastasis.

The activation of Ephrin (Eph) receptors, the largest tyrosine kinase families of cell surface receptor, has recently been addressed in human cholangiocarcinoma (CCA). Therefore, the present study aimed to investigate the role of Eph receptors and its ligands in CCA. Of all 50 cases of human CCA tested, immunohistochemical staining demonstrated that EphB2, EphB4, ephrinB1, and ephrinB2 were 100 % positive in CCA tissues with overexpressions of the above proteins as 56, 56, 70, and 48 % of cases, respectively. High expression of EphB2 was significantly correlated with the metastatic status of patients (P = 0.027). We also found that the high co-expression level of EphB2/ephrinB1 or EphB2/ephrinB2 were significantly correlated with the metastatic status of the patients (P = 0.034 and P = 0.024). Furthermore, we showed that the high co-expression level of EphB4/MVD and ephrinB1/MVD were significantly correlated with the metastasis status of CCA patients (P = 0.012 and P = 0.029). We further demonstrated that the EphB2 suppression using siRNA significantly reduced CCA cell migration by decreasing the phosphorylation of focal adhesion kinase (FAK) and paxillin. In conclusion, the upregulation of EphB2 receptors and its specific ligands (ephrinB1 and ephrinB2) leads to CCA metastasis. Suppression of EphB2 expression as well as inhibition of its downstream signaling proteins might serve as possible therapeutic strategies in human CCA.

BMP-7 blocks the effects of TGF-ß-induced EMT in cholangiocarcinoma.

Epithelial-mesenchymal transition (EMT) is characterized by the loss of epithelial markers and the gain of mesenchymal markers. EMT is believed to be a major mechanism supporting cancer cell metastasis. The activation of EMT can be induced by various types of inflammatory cytokines including transforming growth factor ß (TGF-ß) whereas bone morphogenetic protein-7 (BMP-7) can inhibit this process. In this study, the up-regulation of Twist transcription factor and N-cadherin, mesenchymal marker in CCA tissues, has been demonstrated and it has been found that the high expression of Twist was significantly associated with poor prognosis of CCA patients (P = 0.010). Moreover, CCA samples showing Twist nuclear expression were significantly correlated with the up-regulation of N-cadherin (P = 0.024). These results also showed that the inflammatory mediator TGF-ß induces CCA cell migration, one of the metastatic processes possibly via stimulation of Twist, N-cadherin and vimentin expression. Additionally, it has been shown that BMP-7 inhibits TGF-ß-induced CCA cell migration, through inhibition of TGF-ß-mediated Twist and N-cadherin expressions. These data reinforce the rationale to use BMP-7 as an EMT inhibitor to suppress the progression of CCA and might be a therapeutic approach to improve efficiency for CCA treatment.

Loss of E-cadherin promotes migration and invasion of cholangiocarcinoma cells and serves as a potential marker of metastasis.

Tumor progression is characterized by loss of cell adhesion and increase of invasion and metastasis. E-cadherin, a cell adhesion molecule, is frequently downregulated and has been proposed as an important mediator in epithelial-mesenchymal transition (EMT) in tumors. In this study, we investigated the expression of E-cadherin and its association with cancer invasion and prognosis in cholangiocarcinoma (CCA). Immunohistochemistry results demonstrated a statistically significant association between the positive metastasis status with low E-cadherin protein expression in human CCA tissues (P = 0.04). Statistical trends were identified for low E-cadherin level and shorter survival time (P = 0.08). Targeting the E-cadherin expression in CCA cells with siRNA caused upregulation of vimentin, a mesenchymal marker, and disappearance of the E-cadherin/ß-catenin adhesion complex from cell membranes. Moreover, migration and invasion abilities of the cells were increased under this condition. These findings suggest that reduction of E-cadherin contributes to CCA progression by attenuating the strength of cellular adhesion, which affects motility as well as regulating the expression of EMT-related genes during CCA invasion and metastasis. Thus, E-cadherin can act as a central modulator of tumor cell phenotype and is a potential metastasis marker in CCA.

Milk fat globule epidermal growth factor 8 serves a novel biomarker of opisthorchiasis-associated cholangiocarcinoma.

Milk fat globule epidermal growth factor 8 (MFG-E8) is a pleiotropic secreted glycoprotein to play roles in mediating immune tolerance and homeostasis maintenance and enhancing angiogenesis. To evaluate its value as a biomarker in opisthorchiasis-associated cholangiocarcinoma (CCA), the present study investigated MFG-E8 expression kinetics during the tumorigenesis in Opisthorchis viverrini infection-induced CCA, and demonstrated its expression in the tumor tissues of CCA patients and its serum level among them. During the tumorigenesis of CCA, MFG-E8 expression was increased in a time-dependent manner with the pathological processes. Absolutely higher expression of MFG-E8 messenger RNA was detected in the tumor tissues from CCA patients, compared with those in adjacent tissues. Immunobiochemical analysis showed that more than 90% CCA cases were positive and the positive reaction located in the membrane and cytoplasm of the tumor cells. Moreover, the average serum level in the CCA patients was significantly higher than that in healthy individuals and those with O. viverrini infection or other parasitosis. Correlation analysis of MFG-E8 expression with CCA clinicopathology revealed that a high expression of MFG-E8 protein was significantly bound with a poor differentiation, pathological advanced stage, and metastasis of CCA. The multivariation analysis indicated that MFG-E8 was an independent prognostic factor. In addition, short hairpin RNA-mediated MFG-E8 knockdown in CCA cell line obviously suppressed the cell proliferation. Our results strongly suggested that MFG-E8 is a promising biomarker for the diagnosis, prognosis, and therapy target of opisthorchiasis-associated CCA.

PGE2 signaling and its biosynthesis-related enzymes in cholangiocarcinoma progression.

Prostaglandin E2 (PGE2) involves in progression of various chronic inflammation-related cancers including cholangiocarcinoma (CCA). This study aimed to determine the role of PGE2 signaling, its biosynthesis-related enzymes in a clinical prognosis, and their targeted inhibition in CCA progression. The immunohistochemical staining of cyclooxygenase (COX)-1, COX-2, mPGES-1, EP1, and EP4 was examined in CCA tissues, and their expressions were compared with clinicopathological parameters. The effect of PGE2 on levels of its signaling molecules was examined in CCA cell lines using proteome profiler array. The suppression of mPGES-1 using a small-molecule inhibitor (CAY10526) and small interfering RNA (siRNA) was determined for growth and migration ability in CCA cells. The results indicated that strong expressions of COX-1, COX-2, mPGES-1, EP1, and EP4 were found in CCA tissues as 87.5, 47.5, 52.5, 55, and 80 % of frequencies, respectively. High mPGES-1 expression was significantly correlated with tumor stages III-IV (p = 0.001), lymph node metastasis (p = 0.004), shorter survival (p = 0.009), and prognostic indicator of CCA patients (HR = 2.512, p = 0.041). Expressions of COX-1, COX-2, and EP receptors did not correlate with data tested from patients. PGE2 markedly enhanced protein levels of integrinα6, VE-cadherin, Jagged1, and Notch3, and CAY10526 suppressed those protein levels as well as PGE2 production in CCA cells. CAY10526 and siRNA mPGES-1 markedly suppressed mPGES-1 protein levels, growth, and migration abilities of CCA cell lines. In conclusion, PGE2 signaling strongly promotes CCA progression. Therefore, inhibition of PGE2 synthesis by suppression of its biosynthesis-related enzymes could be useful for prevention and treatment of CCA.